The goal of this project is to understand the regulation of dinitrogenase reductase by reversible ADP-ribosylation. The dinitrogenase reductase protein of the nitrogenase enzyme in Rhodospirillum rubrum is inactivated by ADP{-ribosylation of arg101 in response to ammonium or darkness. DRAT is the ADP-ribosyltransferase responsible for inactivation and DRAG is the glycohydrolase that activates dinitrogenase reductase by removing ADP-ribose. Both DRAT and DRAG are independently regulated in the cell, and the overall goal of this project is to understand the signal transduction pathways that regulate DRAT and DRAG. Structural information for both DRAT and DRAG will be obtained by crystallography, sequence modeling and site-specific chemical modification of the enzymes. This information will be used to direct mutagenesis of genes encoding DRAT and DRAG, and the mutant forms of DRAT, DRAG, and dinitrogenase reductase will be tested for their activities and regulation. NAD serves as the ADP-ribose donor, and the site of NAD binding to DRAT and dinitrogenase reductase will be established. The hypothesis that occupancy of the NAD binding sites regulates DRAT activity will be tested in vitro and in vivo; UV photoaffinity techniques will be developed for the in vivo tests. Binding of NAD to DRAT and to dinitrogenase reductase will be quantified. Effectors of DRAT and DRAG activities will be sought using in vitro assays. Mutant forms of DRAT and DRAG that are unregulated "always active" will serve as controls in these assays. Effectors will be isolated and identified. ADP-ribosylation is increasingly recognized as an important mechanism of regulation in eucaryotes and procaryotes. A number of pathogenic bacteria produce toxins that ADP-ribosylate crucial protein targets in their eucaryotic hosts.